Method for improved diagnosis of dysplasias

ABSTRACT

The present invention relates to a method for improved diagnosis of dysplasias based on simultaneous detection of INK4a gene products and at least one marker for cell proliferation. Particularly the present invention provides a method for discriminating dysplastic cells over-expressing INK4a gene products from cells over-expressing INK4a gene products without being dysplastic by detection of a marker suitable for characterising the proliferation properties of the respective cell. The characterisation of the proliferation properties may comprise the detection of a marker or a set of markers characteristic for active cell proliferation and/or a marker or a set of markers characteristic for retarded or ceased cell proliferation. The method presented herein thus enables for a specific diagnosis of dysplasias in histological and cytological specimens.

This application is a National Stage of International ApplicationPCT/EP03/050738, filed Oct. 21, 2003, published May 6, 2004, under PCTArticle 21(2) in English; which claims the priority of EP 02024030.5,filed Oct. 28, 2002; and EP 03100584.6, filed Mar. 7, 2003.

FIELD OF THE INVENTION

The present invention relates to a method for improved diagnosis ofdysplasias based on simultaneous detection of INK4a gene products and atleast one marker for cell proliferation. In particular, the presentinvention provides a method for discriminating dysplastic cellsover-expressing INK4a gene products from cells over-expressing INK4agene products without being dysplastic by detection of a marker suitablefor characterising the proliferation properties of the respective cell.The characterisation of the proliferation properties may comprise thedetection of a marker or a set of markers characteristic for active cellproliferation and/or a marker or a set of markers characteristic forretarded or ceased cell proliferation. The method presented herein thusenables a specific diagnosis of dysplasias in histological andcytological specimens.

The detection of the over-expression of p16^(INK4a) in biologicalsamples has proven as a useful marker in the detection of anogenitallesions, such as carcinoma of the uterine cervix (see WO00/01845; Klaeset al., Int. J. Cancer: 92, 276-284 (2001)). The method based onp16^(INK4a)-specific immuno-chemical staining allows for a sensitive andspecific identification of dysplastic cells in tissue sections and incytological samples.

In immuno-histochemical examinations of tissues, dysplastic andneoplastic cells can be stained using a p16^(INK4a) specific antibodymediated staining procedure. The histological diagnosis of neoplasticlesions can thus be supported by a staining procedure based on amolecular marker characteristic for transformation of cells inanogenital lesions. The diagnosis, whether or not cells are neoplastic,in these procedures is not solely based on the p16^(INK4a) specificstaining, but does also rely on the histological information.

This is due to the fact that, in about 20-30% of samples, metaplasticcells show some immuno-reactivity with p16^(INK4)a specific antibodies,and thus are stained in the course of the procedures. Yet the stainingpattern of these metaplastic cells differs from the pattern ofneoplastic lesions.

Metaplastic cells give rise to a patchy or focal staining pattern,whereas neoplastic lesions give rise to diffuse staining pattern.Moreover, the staining intensities of metaplastic cells arepredominantly less than that of neoplastic cells.

The common methods used in screening tests for the early detection ofdysplasias and/or neoplasias do not employ histology based tests, butrather rely on cytological testing procedures. Yet especially in caseswhen there is no histological information available concerning thearchitecture of tissues, such as, for example, in cytologicalexaminations, testing for p16^(INK4a) over-expression alone may lead tofalse positive results. This is due to the fact that those fractions ofmetaplastic cells expressing p16^(INK4a) at detectably elevated levelsmay not be differentiated by means of a histologic criteria.

The percentage of cells showing over-expression of p16^(INK4a) increasesin the course of emergence of dysplasias. So, in neoplastic orpre-neoplastic stages, when only a restricted population of neoplasticor pre-neoplastic cells is present in samples, the immunoreactivity ofp16^(INK4a) may be weak. This weak immuno-reactivity may be of about thelevel as the level caused by metaplastic cells. In later stages ofdysplasias, the overall immunoreactivity of p16^(INK4a) is stronger, soneoplastic lesions are easily discernible from metaplasias even in acytological testing format. This might lead to cases where the presenceof metaplastic cells expressing p16^(INK4a) might be confused with thepresence of neoplastic cells, and thus produce a false positive result.

Especially in screening tests, where the detection of early stages ofneoplasias is desirable, this condition is quite unpleasant. This isespecially true, as the p16^(INK4a) based diagnosis has proven to be avaluable tool in histological examinations and the application incytological based screening procedures would be able to enhance theseestablished procedures.

To reduce false positive results in cytological testing formats andthereby further enhance the fidelity of the p16^(INK4a) mediateddiagnosis of anogenital lesions, a method for discriminating themetaplasias from neoplastic and dysplastic lesions would be desirable. Amethod for the discrimination of metaplasias from neoplastic andpre-neoplastic lesions is provided within the embodiments claimedaccording to the present invention.

For supporting the discrimination of metaplasias from neoplastic lesionsin testing procedures based on the over-expression of p16^(INK4a), amarker molecule would be desirable that is expressed in neoplasticand/or pre-neoplastic cells and tissues and which is not expressedsimultaneously with INK4a gene products in one single metaplastic cell.

A solution to the problem present in the art is provided by the methodsclaimed according to this invention. In the course of the experimentsleading to the present invention, the inventors have found that acombination of detection of the presence or absence and/or the level ofp16^(INK4a), in combination with at least one marker for cellproliferation, such as e.g. Ki67, Ki-S2, mcm5 or mcm2, may solve theproblem present in the art.

In the art, a couple of documents pertaining to the use of a combinationof molecular markers for improved diagnosis of dysplasias is presented.In WO0208764, a method for improved diagnosis of cervical malignanciesis disclosed using a combination of an HPV marker and a marker for cellproliferation or viral activity. p16^(INK4a) is mentioned in the contextof this invention as a viral activity marker to be combined with HPVmarkers.

In EP1217377, a method for automated detection of cervical malignanciesis disclosed that is mediated by detection of more than one markermolecules. Some defined marker combinations are named within thedocument. There is no disclosure in this document relating to the choiceof suitable markers for a combination. The purpose of the combination inthis application is improved fidelity of the automated analysis ofstaining patterns in biological cytological specimens. This documentmentions combination of p16^(INK4a) with other tumor markers.

WO02059616 discloses a method for detection of cell-cycle disturbancesfor improved diagnosis of cervical malignancies. The document disclosesthat dysplastic cells exhibit disturbances in cell cycle control, andmay thus be identified by means of detection of cyclin E type proteinstogether with post G1 substances in cells.

Jeffrey Keating in “Ki67, Cyclin E, and p16^(INK4a) Are ComplimentarySurrogate Biomarkers for human papilloma Virus-Related CervicalNeoplasia” (American Journal of Surgical Pathology 25(7): 884-891,(2001)) discloses the complementarity of the use of p16^(INK4a), Ki67,and Cyclin E in the course of diagnosis of cervical dysplasias. Thedocument refers to the problems of each single marker in the detectionof dysplasias, and states that p16^(INK4a) in combination with Cyclin Emay be suitable to overcome the drawbacks of the single markermolecules, especially when cytological specimens are under examination.No disclosure teaching use of p16^(INK4a) in concert with a markercharacteristic of proliferating cells such as Ki67 is given. Thedocument does not teach the combination of p16^(INK4a) for use indiagnostic methods; moreover, the disclosure pertains to a restricteduse of Ki67 in cervical differential diagnosis, and thus, teaches awayfrom the use of this marker in diagnosis of cervical malignancies.

In the art, there is no disclosure teaching the use of a combination ofp16^(INK4a) with a marker characteristic of cell proliferation for usein a diagnostic method for improved discrimination of p16^(INK4a)positive non-dysplastic cells from p16^(INK4a) positive dysplasias.WO02059616 does not give a hint as to the use of p16^(INK4a) in thedetection of dysplastic cell proliferation. EP1217377 does not teach apurpose for the combination of tumor markers in the course of thedetection other than the automation of the analysis process. There is nodisclosure pertaining to the advantage of combinations for specificdiscrimination purposes such as discrimination of dysplastic cells frome.g. metaplastic cells in cervical specimens.

SUMMARY OF THE INVENTION

The inventors of the present invention sought to overcome the drawbackpresent in the art that p16^(INK4a), over-expressed in variousdysplasias, may also be detected in some other non-dysplastic cells. Thediscrimination between non-dysplastic p16^(INK4a) positive cells anddysplastic cells over-expressing p16^(INK4a) may be based on theproliferation characteristics of the respective cells. In normal controlcells, p16^(INK4a) inhibits cdk4, and thus inhibits proliferation. Incontrast, in dysplastic cells, this regulation is impaired. Thus,p16^(INK4a) does not lead—despite its uncommonly high expressionlevel—to an inhibition of the cell proliferation.

The inventors of the present invention found that dysplastic cells maybe discriminated from cells exhibiting control cell proliferation by thesimultaneous detection of p16^(INK4a) with a marker characteristic forcell proliferation. Due to the fact that, in normal cells, elevatedlevels of p16^(INK4a) inhibit cell proliferation, cells over-expressingp16^(INK4a) may be classified as being dysplastic provided they areexhibiting the characteristics of active cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withdrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 1 shows staining of a severe dysplasia usingantibodies directed against p16^(INK4a). For experimental details, seeExample 1. Immunoreactivity for p16^(INK4a) renders green fluorescence.Almost all cells of the lesion are diffusely positive stained in thecytoplasm and in the nuclei.

FIG. 2 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 2 shows staining of a severe dysplasia usingantibodies directed against Ki67. For experimental details, seeExample 1. Immunoreactivity for Ki67 renders red fluorescence. Manycells of the dysplasia show nuclear positivity for Ki67.

FIG. 3 is an example of fluorescent double staining of a histologicalspecimen of the cervix uteri. FIG. 3 shows staining of a severedysplasia using antibodies directed against Ki67 and those directedagainst p16^(INK4a). For experimental details, see Example 1.Immunoreactivity for Ki67 renders red fluorescence, immunoreactivity forp16^(INK4a) renders green fluorescence and the overlay of red and greenfluorescence renders yellow fluorescence. Many cells of the dysplasiashow nuclear positivity for Ki67, as well as positivity for p16^(INK4a),and thus give rise to yellow fluorescence.

FIG. 4 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 4 shows staining of a squamous metaplasiausing antibodies directed against p16^(INK4a). For experimental details,see Example 1. Immunoreactivity for p16^(INK4a) renders greenfluorescence. Some cells of the metaplasia stain diffusely positive inthe cytoplasm and in the nuclei.

FIG. 5 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 5 shows staining of a squamous metaplasiausing antibodies directed against Ki67. For experimental details, seeExample 1. Immunoreactivity for Ki67 renders red fluorescence. Manycells of the squamous metaplasia show nuclear positivity for Ki67. Areasthat have been identified as positively expressing p16^(INK4a) do notshow any positive staining for Ki67, indicating lack of expression ofthe antigen in these areas.

FIG. 6 is an example of fluorescent double staining of a histologicalspecimen of the cervix uteri. FIG. 6 shows staining of a squamousmetaplasia using antibodies directed against Ki67 and againstp16^(INK4a). For experimental details, see Example 1. Immunoreactivityfor Ki67 renders red fluorescence, immunoreactivity for p16^(INK4a)renders green fluorescence and the overlay of red and green fluorescencerenders yellow fluorescence. Areas that are positively expressingp16^(INK4a) do not show any positive staining for Ki67, indicating lackof expression of the antigen in these areas. No cells in the specimengive rise to yellow fluorescence.

FIG. 7 is an example of chromogenic double staining of a histologicalspecimen of the cervix uteri. FIG. 7 shows staining of a severedysplasia using antibodies directed against Ki67 and againstp16^(INK4a). For experimental details, see Example 6. Immunoreactivityfor Ki67 renders red nuclear staining, immunoreactivity for p16^(INK4a)renders brownish staining over the whole cell, and double stainingrenders brown cells with red nuclei. Many cells of the dysplasia shownuclear positivity for Ki67, as well as positivity for p16^(INK4a), andthus, give rise to a pattern of brown cells with red nuclei.

FIG. 8 is an example of chromogenic double staining of a cytologicalspecimen of the cervix uteri. FIG. 8 shows staining of a severedysplasia using antibodies directed against Ki67 and againstp16^(INK4a). For experimental details, see Example 7. Immunoreactivityfor Ki67 renders red nuclear staining, immunoreactivity for p16^(INK4a)renders brownish staining over the whole cell, and double stainingrenders brown cells with red nuclei. Many cells of the dysplasia shownuclear positivity for Ki67 as well as positivity for p16^(INK4a), andthus, give rise to a pattern of brown cells with red nuclei.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for discrimination ofneoplastic, pre-neoplastic and/or dysplastic lesions from non-dysplasticcells showing elevated levels of p16^(INK4a), in biological samples in ahistological or cytological testing procedure based on the detection ofthe presence or absence of cells expressing p16^(INK4a) genesimultaneously with cell proliferation markers in said biologicalsamples. Suitable markers for cell proliferation may be, e.g., Ki67,Ki-S2, Ki-S5, mcm5, or mcm2. In one embodiment of the invention, proteinor mRNA of the markers for cell proliferation may serve as a marker fordiscrimination of metaplasias from early dysplastic or pre-neoplasticlesions in samples.

One aspect of the present invention is to provide a method fordiscriminating dysplastic cells over-expressing INK4a gene products fromother cells expressing INK4a gene products at a detectable level inbiological samples comprising determining in a cytological orhistological testing procedure the co-expression of at least two markermolecules in at least one single cell, wherein at least one markermolecule is an expression product encoded by the INK4a gene and at leastone further marker molecule is a cell proliferation marker, wherein theover-expression of at least one INK4a gene product and expression of atleast one marker for active cell proliferation at an immuno-chemicallydetectable level within said single cell is indicative of the dysplasticstate of the cell, and wherein the over-expression of at least one INK4agene product and expression of at least one marker for senescence,terminal differentiation of cells, apoptosis, or cell cycle arrest at adetectable level within said single cell is indicative of thenon-dysplastic state of the cell.

A second aspect of the present invention relates to a test kit fordetermination of dysplasias in samples according to the method disclosedherein.

During the experiments leading to the present invention, it was foundthat, under certain circumstances, non-dysplastic cells may showimmuno-reactivity for p16^(INK4a). The inventors found that these cellsexhibiting ordered control of cell proliferation in response to theelevated levels of the INK4a gene products are subject to growth arrest.Thus, these individual cells do not show immuno-reactivity for markersof cell proliferation. In contrast, transformed cells over-expressingp16^(INK4a) exhibit dysregulated control of cell proliferation, and donot respond to the elevated level of p16^(INK4a) by cessation ofproliferation. Thus, these dysplastic cell show simultaneous expressionof p16^(INK4a) with markers for cell proliferation. The inventors thusfound that a simultaneous detection of p16^(INK4a) with markers for cellproliferation may serve to discriminate dysplastic cells from arrestedcells over-expressing p16^(INK4a) such as, e.g., metaplastic cells.

The discovery that p16^(INK4a) over-expressed in various dysplasias mayalso be detected in some other non-dysplastic cells led the inventors ofthe presented method to establish a technique for discrimination betweennon-dysplastic p16^(INK4a) positive cells and dysplastic cellsover-expressing p16^(INK4a) based on the proliferation characteristicsof the respective cells. Whereas in normal control cells p16^(INK4a)inhibits cdk4, and thus inhibits cell proliferation, this is not truefor dysplastic cells.

Thus, in dysplastic cells p16^(INK4a) does not lead—despite it'suncommonly high expression level—to an inhibition of the cellproliferation.

The method disclosed herein is based on the fact that dysplastic cellsmay be discriminated from cells exhibiting normal control cellproliferation by the simultaneous detection of an INK4a gene productsuch as, e.g., p16^(INK4a) with a marker characteristic for cellproliferation.

The term marker, as well as marker molecules in general, shall be usedherein to pertain to proliferation marker gene expression products aswell as to INK4a gene expression products.

The denominations given throughout this text for genes may, in part,relate to the genes or proteins as they have been discovered from anyorganism. In the context of the present invention, this denominationshall confer to the respective homologue of the named markers in theorganism which is particularly in question for a method as disclosedherein. In certain embodiments of the present invention, this organismis a mammal and, in one embodiment, may be a human being. Thus, in oneembodiment of the present invention, the named markers shall be thehuman homologues of the respective denominated ones.

Generally throughout the text, the term “(cell) proliferation marker” or“marker for cell proliferation” in the various grammatical forms is usedto denominate proteins as well as nucleic acid markers. In case theprotein name of a marker such as e.g. “replication protein” is usedherein, this use shall be understood metonymically and pertain as wellto the protein as to the nucleic acid marker molecules encoding theparticular protein

A marker useful according to the present invention may be any moleculetranscribed from a gene or any molecule translated from such atranscript. Thus, “gene product”, as used in the context of the presentinvention, may comprise polynucleotides such as, e.g., DNA or RNA andpolypeptides such as proteins, proteoglycans, peptides, etc. “Expressionproduct(s)” as used in the context of the present invention shallcomprise any transcript of a gene locus in forward or reverse directionincluding any reading frames and splicing variants. “Expressionproducts” as used herein shall thus comprise any alternative productsencoded by the nucleic acids of a particular gene locus.

INK4a encoded gene-products, as used in the context of the presentinvention, shall be any mRNA transcribed from the INK4a gene locus orany polypeptide translated from such an mRNA. In one embodiment of theinvention, the expression products encoded by the INK4a gene may exhibitmolecular weights of about 5 to 40 kDa or any value in between, andpreferably of about 10 to 20 kDa or any value in between, and mostpreferably of about 14 to about 19 kDa or any value in between,respectively.

INK4a gene-products suitable for the method according to the presentinvention may comprise, e.g., gene-products such as, e.g., p16^(INK4a)and p14ARF.

The term “(cell) proliferation marker” or “marker for cellproliferation”, as used in the context of the present invention, shallcomprise any marker molecule known in the art to be characteristic forthe proliferation status of cells. The proliferation status may be,e.g., a status of actively proliferating cells, of retarded cellproliferation, of arrested cell proliferation, of senescent cells, ofterminally differentiated cells, of apoptosis, etc. In one embodiment ofthe invention, the cell proliferation marker is a marker moleculecharacteristic for active cell proliferation. In another embodiment ofthe invention, the proliferation marker molecule may be a moleculecharacteristic for arrested, terminally differentiated, senescent, orapoptotic cells.

In certain embodiments, proliferation markers for use in the context ofthe present invention may comprise genes engaged in the DNA replication,such as e.g., proteins of the pre-initiation complex or of thereplication fork. Such molecules may comprise, e.g., helicases, such aseucaryotic helicase or MCM proteins (MCM2, MCM3, MCM4, MCM5, MCM6,MCM7), protein TP as disclosed in WO0050451 and WO0217947 (alsodenominated HELAD1, Pomfil2, Unc-53), kinases or phosphatases engaged inthe replication process such as, e.g., CDC6, CDC7 protein kinase, Dbf4,CDC14 protein phosphatase, CDC45 and MCM10. Furthermore, proliferationmarkers may comprise proteins engaged in the processive replication forksuch as, e.g., PCNA or DNA polymerase delta, replication protein A(RPA), replication factor C (RFC), and FEN1.

In other embodiments, the proliferation markers may comprise moleculesnecessary for the maintenance of cell proliferation such as Ki67, Ki-S5,or Ki-S2. In this embodiment, proteins may be present, e.g., throughoutthe whole cell cycle. They are useful for performing a method accordingto the present invention provided they are characteristic of active cellproliferation and are not significantly expressed in arrested,terminally differentiated, apoptotic, or senescent states of cells.Ki67, Ki-S2, and Ki-S5 as used herein shall denominate the proteinmarker molecules detected by the respective antibodies as well as thenucleic acids encoding these antigens.

In another embodiment, the cell proliferation markers for use in amethod according to the present invention may be a marker moleculecharacteristic of retarded or ceased cell proliferation such as, e.g., asenescence marker, a cell cycle arrest marker, a marker characteristicfor terminally differentiated cells, or an apoptosis marker. Suchmolecules comprise, e.g., p21, p27, Caspases, BAD, CD95, fas-ligand,parp-proteins, etc.

Discrimination as used in the context of the present invention shallcomprise an assessment whether a sample is to be classified in one oranother way. In one embodiment of the invention, the discriminationpertains to the assessment of a tissue, or components thereof, beingdysplastic or being not dysplastic. Thus, the discrimination as usedherein is a judgement about the growth properties of cells in abiological sample.

In one embodiment of the present invention, the discrimination comprisesthe detection of expression of an INK4a gene product simultaneously withthe detection of expression of a marker characteristic for active cellproliferation. In this case, cells co-expressing both marker moleculesare to be classified as being dysplastic.

In another embodiment of the present invention, the discriminationcomprises the detection of an INK4a gene product simultaneously with thedetection of expression of a marker characteristic for arrested, ceased,or retarded cell proliferation. In this case, cells co-expressing bothmarker molecules are to be classified as being non-dysplastic.

In certain embodiments of the present invention, it will be useful todetect the presence or absence and/or the level of more than two markermolecules. In one embodiment, one INK4a gene expression product will bedetected in concert with two or more markers for cell proliferation.This may be useful to enhance the identification of proliferationproperties of the INK4a gene product-expressing cells in samples. Someproliferation markers are restricted to specific phases of the cellcycle or are present in low abundance in cells. Due to this fact, insome cases, the detection of proliferating cells expressing INK4a geneproducts may be improved by detection of two or more proliferationmarkers. In such cases, e.g., one proliferation being expressed duringthe whole proliferative cell cycle may be detected simultaneously bymarkers characteristic for specific cells cycle phases. For example,Ki67, Ki-S5, or Ki-S2 may be detected together with mcm5, mcm2, PCNA,rpA, rfC, etc. In other cases, e.g., proteins engaged in the DNAreplication may be detected together with Ki67, Ki-S5, or Ki-S2. In yetanother case, Ki67 may be detected together with Ki-S2. It must beunderstood that these examples are intended to exemplify combinatorialpossibilities, and shall not be comprehensive, so that various othercombinations of proliferation markers are likewise useful and suitablein the procedure of the method according to the present invention.

As the case may be, combination of two or more cell proliferation markermolecules may be applied in a method as disclosed herein. In anotherembodiment, two or more marker molecules detectable over largerstretches of the cell cycle or even over the whole cell cycle, or in anactively proliferating cell, may be detected in concert in a method asdisclosed herein. A combination of more than one cell proliferationmarker molecules may be useful generally for improving the sensitivityof the detection of the proliferation characteristics of cells.

In certain embodiments of the present invention, a combination mayfurthermore comprise other marker molecules such as senescence markermolecules, markers for arrested cells, markers for terminallydifferentiated cells, markers for apoptotic cells, markers for viralinfection or for viral activity in cells, or cell cycle regulatoryprotein markers. In certain embodiments, in connection with dysplasiasbeing associated with HPV infection, a detection of HPV associatedmarker molecules or markers for viral activity may be of use for adetection of a dysplasia. The methods useful for detection of detectionof HPV infection in samples are known to those of skill in the art.These methods may comprise methods employing probes specific for HPVagents or may employ nucleic acid amplification reactions. The detectionof a viral infection may be carried out simultaneously or subsequentlyto the detection of the INK4a and proliferation marker molecules.

Dysplastic, as used in the context of the present invention, shall referto dysplasias from mild to severe dysplasias and their precursorystages, as well as carcinoma such as carcinomas in situ or invasivecarcinomas and disseminated tumor cells. Thus, dysplastic as used hereinshall also comprise early and precursory stages of dysplasias andcarcinomas.

Cells over-expressing INK4a gene products without being dysplastic(non-dysplastic as used herein) as mentioned herein may comprise, e.g.,metaplastic cells, senescent cells, terminally differentiated cells, orcells that in certain stages of the cell cycle exhibit elevated levelsof the INK4a gene products. In certain cells, elevated levels of INK4agene products may even be effected as a response to external signalssuch as hormones, transmitters, etc. In one embodiment of the presentinvention, the non-dysplastic cells over-expressing INK4a gene productscomprise, e.g., metaplastic cells, endometrial cells, etc.

The method for detection of the expression level of the INK4a encodedgene-products and/or the proliferation marker gene products according tothe present invention is any method, which may (but need not) be, e.g.,suited to detect even very small amounts of specific biologicalmolecules in biological samples. The detection reaction according to thepresent invention is a detection either on the level of nucleic acids oron the level of polypeptides.

A marker molecule is said to be detectable as used in the context of thepresent invention, provided the marker may be detected in the course ofsuitable detection procedure such as, e.g., in situ hybridization,immuno-chemical staining, hybrid capture assay, etc. The level ofexpression of a marker molecule may be made detectable using suitablereporter reactions such as, e.g., a chromogenic or fluorescence basedimmuno-chemical staining or in-situ-hybridization procedure formicroscopic or automated analysis. Suitable methods for enhancing thereporter signal known to those of skill in the art may be applied in thecourse of a method according to the present invention. Thus, the markeris said to be detectable in a case where the staining supersedes therespective background staining inherently obtained in theimmuno-chemical staining procedure so as to produce significant stainingresults.

The marker molecules may be detected using reagents that specificallyrecognize these molecules. The detection reaction for the INK4agene-products and/or the proliferation marker gene products may compriseone or more reactions with detecting agents either recognizing theinitial marker molecules or recognizing the prior molecules used torecognize other molecules.

In certain embodiments of the present invention, two or more probes maybe used for the detection of one single marker molecule. For example,two or more different binding agents (e.g., antibodies) oroligonucleotide probes directed against one single marker molecule (or,as the case may be, against different epitopes or different sequences)may be used in the course of the method as disclosed herein.

The detection of the different gene products may be performed in onereaction vessel or container or in different containers simultaneouslyor subsequently in time. Thus, the different gene products may bedetected simultaneously in one cell co-expressing both products.Otherwise, cells co-expressing the gene products may be used forseparated detection reaction (separated in space or in time) to detecteach a single marker in the cells. In another embodiment, there might becells expressing one or the other marker. The detection of the markermolecules in the different cells may also be performed simultaneously orseparately in time and/or space.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the markermolecule gene-products. The reporter reaction may be, for example, areaction producing a coloured compound, a bioluminescence reaction, afluorescence reaction, generally a radiation emitting reaction, etc.

In certain embodiments, different marker molecules may be recognized byagents that produce different reporter signals, so that the signalsreferring to marker molecules can be distinguished. In one preferredembodiment of the invention, the detection of the expression of the twoor more INK4a gene-products and/or proliferation marker gene products iscarried out simultaneously. In this case, the reporter reaction mayemploy, for example, different fluorescent labels for the differentmolecules detected.

However, within the context of the present invention, it must notnecessarily be answered whether the one or the other proliferationmarker or INK4a marker gene product is expressed in the cells. Incertain embodiments, the question is whether any proliferation markerand/or INK4a gene product is expressed. In the course of theexperiments, a procedure may be chosen that gives the same fluorescencesignal as an indication of the presence of a proliferation marker. Thisprocedure is suitable to improve sensitivity of the detection of thecell proliferation characteristics (different markers characteristic foractive cell proliferation). As the case may be, the procedure may beapplied so as to render one detectable signal for three, four, or evenmore marker molecules characteristic for cell proliferation.Analogously, the same may under certain circumstances be true for theINK4a gene expression products. It must be understood that differentstaining signals for different proliferation marker molecules may bedesirable. The procedures may be applied to the necessities of therespective experiment.

In certain embodiments of the present invention, a combination of one ormore (e.g., two different) INK4a gene products may be detected with acombination of one or more (e.g., a set of two, a set of three, a set offour, a set of five or a set of even more) markers for cellsproliferation. In some cases, the detection of the marker molecules forcell proliferation may render only one reporter signal. In other cases,each single marker for cell proliferation may render a specific reportersignal or groups of marker molecules may render specific reportersignals.

Signals for the indication of the presence of immuno-reactivity may bechromogenic signals produced by various methods known in the art.Alternatively, or even in combination, fluorescent signals may be used.Suitable reporter signals comprise fluorescent labels such asfluorescein, rhodamine, etc.

Applicable formats for the detection reaction according to the presentinvention may be blotting techniques, such as Western-Blot,Southern-blot, Northern-blot, and immunocytochemical, orimmunohistochemical procedures. The blotting techniques are known tothose of ordinary skill in the art, and may be performed as, for exampleelectro-blots, semidry-blots, vacuum-blots or dot-blots.Immunocyto/histochemical staining procedures are known to those of skillin the art, and may comprise binding agent-mediated detection ofpolypeptides as well as in situ hybridisation techniques. Both differenttechniques may even be applied simultaneously. In certain embodiments,hybrid capture of nucleic acids may be used for the detection.Amplification reactions may also be applicable for the detection of e.g.nucleic acid molecules.

In one embodiment of the invention, the detection of the level of INK4aand/or proliferation marker gene-products is carried out by detection ofthe respective nucleic acids (e.g., mRNA) or fragments thereof presentin the sample. The means for detection of nucleic acid molecules areknown to those skilled in the art. The procedure for the detection ofnucleic acids can be carried out by, for example, a binding reaction ofthe molecule to be detected to complementary nucleic acid probes,proteins with binding specificity for the nucleic acids, or any otherentities specifically recognizing and binding to said nucleic acids. Inone embodiment, in situ hybridisation of oligonucleotide probes tonucleic acids in a sample may be used for the detection of expressionproducts or markers.

Probes as used in the context of the present invention may be any agentbinding specifically to a molecule. In the case of nucleic acids, aprobe may be an oligonucleotide hybridising to a particular sequence. Inone embodiment, the probe may be, e.g., a primer. In the case of thedetection of polypeptides or proteins, the probe as used herein may be,e.g., a binding agent such as an antibody. In certain embodiments of thepresent invention, the probes may be detectably labelled. The label maybe selected from the group comprising a radioisotope, a bioluminescentcompound, a chemiluminescent compound, a fluorescent compound, a metalchelate, or an enzyme. Probes may be applied in any detection procedureknown in the art as, for example, in the course of an in situhybridisation procedure, in the course of hybrid capture assays, in thecourse of immuno-chemical staining reaction, in the course of blottingtechniques, etc.

This method may be performed as well in vitro as directly in situ forexample, in the course of a detecting staining reaction. Another way ofdetecting the marker mRNAs in a sample performed in the method accordingto the present invention is an amplification reaction of nucleic acids,which can be carried out in a quantitative manner such as, for example,the polymerase chain reaction. In a preferred embodiment of the presentinvention, real time (RT) PCR may be used to quantify the level ofmarker mRNAs in samples of dysplasias or tumors (cells or tissuesamples).

In another preferred embodiment of the invention, the detection of thelevel of INK4a and/or proliferation marker gene-products is carried outby determining the level of expression of a protein or fragmentsthereof. The determination of the marker gene-product on the proteinlevel can, for example, be carried out in a reaction comprising abinding agent specific for the detection of the particular markerpolypeptide.

The binding agents can be used in many different detection techniquesincluding, for example, in western-blot, ELISA or immuno-precipitation.Generally, polypeptide binding agent based detection can be carried outas well in vitro as directly in situ as, for example, in the course ofan immuno-chemical staining reaction. Any other method for determiningthe amount of particular polypeptides in biological samples can be usedaccording to the present invention.

The immuno-cytochemical imaging procedures for use in the context of thepresent invention may comprise, e.g., the staining of cytological orhistological preparations with chromogenic or fluorescent dyes. Thestaining may comprise, e.g., binding of the molecules to be detected bya first binding agent, which itself is detected by a secondary bindingagent which may be labelled. The first binding agent may in certainembodiments be a nucleic acid or a protein binding agent (e.g. anantibody) and the secondary binding agent may be, e.g., a secondaryantibody recognizing the first binding agent.

Any methods known in the art for performing staining of cytochemical orhistochemical staining may be applied in the course of a methodaccording to the present invention.

Binding agents as used in the context of the present invention for thedetection of the level of INK4a polypeptides such as p16^(INK4a) orp14ARF polypeptides and proliferation marker polypeptides such as, e.g.,mcm5, mcm2, Ki67, Ki-S5, PCNA, or Ki-S2 polypeptides may compriseantibodies and antigen-binding fragments, bi-functional hybridantibodies, peptidomimetics containing minimal antigen-binding epitopes,anti-cullines (anti-caline™), etc.

An antibody or antigen-binding agent is said to react specifically if itreacts at a detectable level with a protein disclosed herein, and doesnot significantly react with other proteins. The antibodies according tothe present invention may be monoclonal or polyclonal antibodies. Asused herein, the term antibody or monoclonal antibody is meant toinclude intact molecules as well as antibody fragments. Moreover,antibodies of the present invention include chimeric, single chain, andhumanized antibodies.

According to the present invention binding agents may be used isolatedor in combination. By means of combination, it is possible to achieve ahigher degree of sensitivity. The term antibody, preferably, relates toantibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations.

Monoclonal antibodies are made from antigen-containing fragments of thepolypeptide of the invention using any of a variety of techniques knownto those of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In one such technique, an immunogen comprising the antigenicpolypeptide, or a synthetic part thereof, is initially injected into anyof a wide variety of mammals (e.g., mice, rats, rabbits, sheep, andgoats). In this step, the polypeptides of this invention may serve asthe immunogen without modification. Alternatively, particularly forrelatively short polypeptides, a superior immune response may beelicited if the polypeptide is joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. The immunogen isinjected into the animal host, preferably according to a predeterminedschedule incorporating one or more booster immunizations, and theanimals are bled periodically. Polyclonal antibodies specific for thepolypeptide may then be purified from such antisera by, for example,affinity chromatography using the polypeptide coupled to a suitablesolid support.

Within the context of the present invention, it must not necessarily beanswered whether one or the other proliferation marker is expressed inthe cells. In certain embodiments, the main question may be whether anyproliferation marker is expressed. In the course of the experiments, aprocedure was chosen that gives the same fluorescence signal as anindication of the presence of a proliferation marker. This procedure issuitable to improve sensitivity of the detection of the cellproliferation characteristics. As the case may be, the procedure may beapplied so as to render one detectable signal for three, four, or evenmore marker molecules characteristic for cell proliferation.Analogously, under certain circumstances, the same may be true for theINK4a gene expression products. It must be understood, that, as the casemay be, different staining signals for different proliferation markermolecules may be desirable. The procedures may be applied to thenecessities of the respective experiment.

The INK4a gene-products and/or proliferation marker gene products may,according to the present invention, be detected simultaneously. In thiscontext, simultaneously according to the present invention shall meaneither literally at the same instant or within the same testingprocedure, whereby the single detection steps are temporarilyconsecutive.

The detection procedure according to the present invention may,furthermore, comprise a cytochemical staining procedure rendering achromogenic or fluorescent staining of cells or cell compartments. Suchstaining procedures are known to those of skill in the art and maycomprise, for example, staining for acidophilic or basophilicstructures, of subcellular regions (e.g. the nucleus, the mitochondria,the golgi, the cytoplasm, etc.), of specific molecules (the chromosomes,of lipids, of glycoproteins, of polysaccharides, etc.) in thecytological specimens. Fluorescence dyes such as DAPI, Quinacrin,Chromomycin, etc. may be employed. Furthermore, chromogenic dyes such asAzan, Acridin-orange, Hematoxylin, Eosin, Sudan-red, and Thiazin-stains(Toluidin-blue, Thionin) may be applied. In other embodiments, stainingprocedures such as Pap-staining, Giemsa-staining, Hematoxylin-Eosinstaining, van-Gieson staining, Schiff-staining (using Schiff reagent),staining procedures employing precipitation of metals (such as, e.g., ofsilver in staining procedures employing Silver Nitrate), or insolublestains such as, e.g., of Turnbulls-blue (or other insoluble metalcyanides), etc. may be used in the course of a method as disclosedherein. It must be understood, that the named dyes and staining methodsshall be examples for the applicable methods, and that any other methodknown in the art may be applied to a method as disclosed herein.

The staining procedures may produce chromogenic stains for lightmicroscopic inspection or fluorescent stains for inspection underfluorescence microscopic conditions. In another embodiment of thepresent invention, radiation emitting procedures, procedures employingsubstances impairing the transmission of radiation, or other contrastmedia for imaging of the cytological conditions in a sample (e.g., thegeneration of optical impression by means such as(micro-)autoradiographic or (micro-)radiographic picture generation) maybe of use for a method according to the present invention.

All the staining and imaging procedures may be used for analysis notonly in microscopic procedures, but also in automated analysisprocedures such flow cytometry, automated microscopic (computerized orcomputer aided) analysis, or any other method for analysis of stainedcytological specimens.

The analysis of the staining or imaging results of the differentprocedures may be performed in a single analysis step or in differentsubsequent steps. For example, the light microscopic inspection of aspecimen may be performed before or after fluorescence microscopicinspection of the specimen. In fluorescence microscopy, the analysis ofdifferent stains with different excitation wavelengths may be analyzedsimultaneously or subsequently. Other imaging methods may be employedsimultaneously or subsequently to the named procedures.

There may be various circumstances, under which combinations ofdifferent staining methods will be suitable. For example, in cases whereno satisfying cytological staining results may be achieved byimmuno-chemical staining, the additional application of generalcytological staining techniques may be suitable.

A sample according to the method of the present invention may compriseany sample comprising cells. Samples may comprise, e.g., secretions,smears, body fluids, and cell- or tissue-samples.

In one embodiment of the present invention, samples comprise cells ofthe anogenital tract, of the respiratory tract, or of the skin and itsappendages. In certain embodiments, the cells may be cells of theuterine cervix, the vagina, the vulva, the penis, the anus, the rectum,the bronchic tree, the lung, the peritoneum, the peritoneal space, thenaso-pharyngeal space, the oral cavity, or the skin. In certainembodiments of the present invention, the sample may be a histologicalsample, a biopsy, or a cytological sample such as, e.g., a smear, aswab, a wash, or a body fluid containing cells (sputum, a secretion,saliva, etc.). In certain embodiments of the present invention, thesamples may comprise cells infected by papilloma virus. The samples may,in certain embodiments, comprise cervical smears, bronchioalveolarlavages, etc.

In certain special embodiments of the present invention, the sample maybe prepared as a monolayer or thin layer preparation of a cytologicalspecimen. The respective methods for preparation of monolayer orthin-layer preparation in cytology are known to those of skill in theart. In one embodiment, the preparation may comprise, e.g., the ThinPreptechnology. Other methods comprise conventional smears, or methodemploying suspensions of cells for preparation of the cytologicalspecimens.

Preparation of a sample may comprise, e.g., obtaining a sample of atissue, of a body fluid, or of cells from a patient. According to thepresent invention, preparation of the sample may also comprise severalsteps of further preparations of the sample, such as preparation ofdissections, preparation of cell suspensions, spreading or applying thecells to be examined onto microscopic slides, preparation of tissuearrays, isolation of polypeptides or nucleic acids, preparation of solidphase fixed peptides or nucleic acids, or preparation of beads,membranes, or slides to which the molecules to be determined are coupledcovalently or non-covalently.

In certain embodiments of the present invention, the method may beperformed in an automated manner. The automation of the method may beachieved by automated staining and analysis of histological orcytological specimens on a solid surface by microscopic means. Inanother embodiment, the automation may comprise a flow-cytometricanalysis of the staining of cells in solution.

The dysplastic lesions to which the method according to the presentinvention may be applied comprise any dysplastic lesions characterizedby over-expression of INK4a gene products such as, e.g., p16^(INK4a) orp14ARF. In certain embodiments, those lesions are dysplasias associatedwith infections by papilloma viruses such as, e.g., HPV. In oneembodiment, the HPV may be a high risk HPV subtype such as HPV16, HPV18,HPV31, HPV 33, HPV35, HPV 39, HPV 45, HPV 51, HPV 52, HPV56, HPV 58, HPV59, HPV 66, HPV 68, etc. In one embodiment, the dysplastic lesions thatmay be detected according to the present invention comprise anogenitallesions, lesions of the respiratory tract, lesions of the head and theneck, or lesions of the skin and its appendages. Such lesions maycomprise dysplasias, e.g., of the anus or rectum, of the vulva, thevagina, the cervix or the penis, of the bronchic tree, the lung, theoral cavity, or the nasopharyngeal space.

Another aspect of the present invention is a testing kit for performingthe method according to the present invention. The kit may be, forexample, a diagnostic kit or a research kit.

A kit according to the present invention comprises at least an agentsuitable for detecting the INK4a gene-products.

Thus a kit according to present invention may comprise:

-   -   reagents for the detection of one or more INK4a gene-products;    -   reagents for the detection of one or more proliferation marker        gene-products;    -   reagents and buffers commonly used for carrying out the        detection reaction, such as buffers, reporters reactants (dyes,        etc.), carrier substances, and others;    -   INK4a gene product samples for carrying out a positive control        reaction; and    -   Proliferation marker gene product samples for carrying out a        positive control reaction.

The reagents for the detection of the marker gene-products may includeany agent capable of binding to the marker gene-products. Such reagentsmay include proteins, polypeptides, nucleic acids, peptide nucleicacids, glycoproteins, proteoglycans, polysaccharides, or lipids.

The INK4a gene-product and proliferation marker gene product samples forcarrying out a positive control may comprise, for example, nucleic acidsin applicable form, such as solution or salt, peptides in applicableform, tissue section samples, or positive cells.

In a preferred embodiment of the invention, the detection of the markergene-products is carried out on the level of polypeptides. In thisembodiment, the binding agents may be, for example, antibodies specificfor the marker gene-products or fragments thereof.

In another embodiment of the test kit, the detection of the markergene-products is carried out on the nucleic acid level. In thisembodiment of the invention, the reagent for the detection may be, forexample, a nucleic acid probe or a primer reverse-complementary to saidmarker nucleic acids.

The present invention provides a method for the discrimination ofneoplastic and/or dysplastic and pre-neoplastic lesions, identifiable byassessment of the over-expression of p16^(INK4a), from other cellscells, which also detectably express p16^(INK4a), in the course ofhistological and/or cytological testing procedures. The method is basedon the detection of expressed gene-products of two or more INK4a geneproducts.

Thus the problem to be solved was to provide a method for discriminationbetween dysplastic cells and other cells lacking malignant growthpotential. The method may be applied to any stage of dysplasias and maybe especially useful in early stages, when cytological diagnosticmethods based on the p16^(INK4a) over-expression need furtherinformation for the identification of metaplastic cells.

Furthermore the present invention provides a kit for performing themethod according to the present invention.

EXAMPLES

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein. For the purpose of illustration, the methods disclosed hereinare exemplified using histological preparations. The histologicalexamples aid to judge whether the cells stained in one or the other wayare to be classified as dysplastic or metaplastic. The methods mayeasily be transferred to cytological specimens by altering the protocolin appropriate manner. These alterations are known to those of ordinaryskill in the art.

Example 1 Immunofluorescent Detection of the Over-Expression ofp16^(INK4a) and Ki67 in Samples of the Uterine Cervix (Double Staining)

Sections of formalin fixed, paraffin embedded tissue samples of thecervix uteri were immunofluorescent stained using antibodies specificfor p16^(INK4a) and Ki67.

The tissue sections were rehydrated through incubation in xylene andgraded ethanol, and transferred to Aqua bidest. Antigen retrieval wascarried out with 10 mM citrate buffer (pH 6.0) for p16^(INK4a) and Ki67.Therefore, the slides were heated in a waterbath for 40 min at 95-98° C.The slides were cooled down to RT for 20 minutes, and transferred towashing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20/DakoCytomation: code no.: S3006).

To avoid non-specific binding of the secondary antibody (species: goat),the specimens were incubated with 10% goat serum for 30 min at RT.

The slides were then incubated with the primary antibodies, mouseanti-human p16^(INK4a) antibody (3.48 μg/ml) and rabbit anti Ki67 (1:25)for 30 min at RT, after which the slides were rinsed with washing bufferand placed in a fresh buffer bath for 5 min. Excess buffer was tappedoff, and each specimen was covered with 200 μl of the secondary reagentcontaining goat anti-mouse antibody, AlexaFluor®488 conjugated and goatanti-rabbit antibody, and Alexa Fluor®546 conjugated, and then incubatedfor 30 min at RT. Then slides were washed two times as before anddirectly mounted with a special mounting medium for fluorescence.

Microscopic examination of the slides revealed that cells immunoreactivewith p16^(INK4a) and also immunoreactive for Ki67 were only found insamples microscopically identified as samples of dysplastic lesions.Cells stained by the p16^(INK4a) specific reaction originating frommetaplasias were not stained by the reaction specific for Ki67.Microscopic inspection of the cell proliferation marker staining showedthat metaplastic cells over-expressing p16^(INK4a) were notimmunoreactive with the antibodies directed against Ki67. Samplescontaining dysplastic tissue areas, in contrast, comprised cells thatwere immunoreactive with KI67 and with antibodies directed againstp16^(INK4a). So, in contrast to dysplasias in metaplasias, no cells weredouble stained using the Ki67 and p16^(INK4a) specific antibodies.

FIGS. 1-6 show the staining results for a severe dysplasia of the cervixuteri and for a squamous metaplasia using antibodies directed againstKi67 and against p16^(INK4a). Immunoreactivity for Ki67 rendered redfluorescence, immunoreactivity for p16^(INK4a) rendered greenfluorescence, and overlay of red and green fluorescence rendered yellowfluorescence. In the dysplastic specimen (FIGS. 1-3), many cells of thedysplasia showed nuclear positivity for Ki67, as well as positivity forp16^(INK4a), and thus gave rise to yellow fluorescence (see FIG. 3). Incontrast, in the metaplastic specimen (FIGS. 4-6), areas that positivelyexpressed p16^(INK4a) did not show any positive staining for Ki67,indicating lack of expression of the antigen in these areas. No doublestaining was observed in this specimen (FIG. 6) and thus, no cells inthe specimen gave rise to yellow fluorescence.

These results show that the double staining of cells with reagentsspecific for Ki67 allows discrimination of p16^(INK4a) over-expressingmetaplasias from dysplasias.

Example 2 Detection of Cells Co-Expressing p14ARF and mcm2 in Samples ofthe Uterine Cervix by In Situ Hybridization

Smears of the uterine cervix may be semi-quantitatively analysed for themRNA level of p16^(INK4a) and mcm2 in an in situ staining reaction. Thestaining reaction is performed as follows:

For rehydration, the spray-fixed smears are incubated in fresh 50% EtOHon a rocking device. The PEG film produced by the fixation procedure isremoved by intensive rinsing. Then, the smears are rinsed in Aquabidest. The smears are incubated with proteinese K (10 μg/ml in PBS) for10 min at 37° C. Then, the slides are transferred to washing buffer(PBS/0.1% Tween20) and finally, the area containing the cells issurrounded with a lipid-pencil.

The hybridization mixture is prepared by mixing 50 μl of ready-to-usehybridization buffer (DAKO A/S, Glostrup, Danmark) with about 5-10 pmolof the probes. The probes are biotin-and Digoxygenin-labelledoligonucleotides of sequences complementary to the respective mRNAs.

The hybridization mixture is heated to 95° C. and afterwards,equilibrated to 37° C. After the boiling procedure, the smears areincubated with each 50 μl of the hybridization mixture for 4 hours at42° C. The samples are washed in excess volumes of the wash buffers twotimes in 2×SSC at 37° C. for 15 min, and once in 1×SSC at 37° C. for 15min. Then, the smears are rinsed two times at room temperature in 2×SSC.Following this washing procedure, the dissections are incubated for 30min with blocking buffer (NEN, blocking buffer) at room temperature,followed by a 1 hour incubation with a 1:100 diluted (in blockingbuffer, see above) Streptavidin-alkaline phosphatase and monoclonalmouse HRP-labeled anti-Digoxygenine antibodies (Molecular Probes). Thesmears are then washed 2 times in 1×PBS/0.1% Triton X-100 for 10 min atroom temperature, followed by one wash step with 1×PBS, 50 mM MgCl₂ (pH9.2) for 10 min at room temperature. Then the staining reaction isperformed with ELF 97 phosphate (Molecular Probes) for 10 sec to 7 minat room temperature. Excess substrate is washed 3 times with 1×PBS/0.1%Triton X-100 for 10 min at room temperature. In a second staining step,the section is incubated with Tyramides-Alexa-Fluor 594 for 10 sec to 7min. Excess substrate is washed 3 times with 1×PBS/0.1% Triton X-100 for10 min at room temperature. Finally the smears are dipped in H₂O_(dest)and embedded with Fluorescence mounting medium (DakoCytomation). Thenthe stained dissections can be analysed by fluorescence microscopy.

Microscopic examination of the slides reveals that cells positive forexpression of p16^(INK4a) and mcm2 are only found in samples that aremicroscopically identified as samples of dysplastic lesions. Cellsstained by the p16^(INK4a) specific reaction that are identifiable asmetaplasias are not stained by the reaction specific for mcm2. Themicroscopic inspection of the mRNA hybridization shows that metaplasticcells over-expressing p16^(INK4a) do not significantly express mRNA ofmcm2. Dysplastic cells, in contrast, are stained by in situhybridization with probes specific for mcm2, and with probes directedagainst p16^(INK4a). So, in contrast to dysplastic cells, in metaplasticcells, no double staining using the Ki67 and p16^(INK4a) specific probesis observed.

These results show that the double staining of cells with reagentsspecific for mcm2 allows discrimination of p16^(INK4a) over-expressingmetaplasias from dysplasias.

Example 3 Immunofluorescent Detection of the Over-Expression ofp16^(INK4a) and Ki-S2 in Samples of the Uterine Cervix (Double Staining)

Merckofix®-fixed cytological samples (conventional smears andliquid-based cytology (ThinPreps®)) of the cervix uteri areimmunofluorescent stained using antibodies specific for p16^(INK4a) andKi-S2.

Conventional smears and liquid based cytological samples (ThinPreps®)are rehydrated in ethanol (50%) for 10 min and transferred in Aquabidest. Antigen retrieval is carried out with 10 mM citrate buffer (pH6.0) for p16^(INK4a) and Ki67. Therefore, the slides are heated in awaterbath for 40 min at 95°-98° C. The slides are cooled down to RT for20 minutes, transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl,0.05% Tween 20/DakoCytomation: code no.: S3006), and finally, thesamples are surrounded with a lipid-pencil.

To avoid non-specific binding of the secondary antibody (species: goat)the specimens are incubated with 10% goat serum for 30 min at RT.

The slides are then incubated with the primary antibodies, mouseanti-human p16^(INK4a) antibody (clone E6H4) (3.48 μg/ml) and rabbitanti-Ki-S2 (1:25) for 30 min at RT, and then the slides are rinsed withwashing buffer and placed in a fresh buffer bath for 5 min. Excessbuffer is tapped off, and the specimen is covered with 200 μl of thesecondary reagent containing goat anti-mouse antibody, AlexaFluor®488conjugated and goat anti rabbit antibody, and Alexa Fluor®546 conjugatedand then incubated for 30 min at RT. Then, slides are washed two timesas before and directly mounted with a special mounting medium forfluorescence.

Microscopic examination of the slides reveals that cells immunoreactivewith p16^(INK4a) and Ki-S2 may be identified microscopically asdysplastic cells. Cells stained by the p16^(INK4a) specific reaction,which are not stained by the reaction specific for Ki-S2, may beclassified by an experienced pathologist as either being metaplastic orof endometrial origin. Microscopic inspection of the cell proliferationmarker staining shows that metaplastic cells over-expressing p16^(INK4a)are not immunoreactive with the antibodies directed against Ki-S2.Dysplastic cells, in contrast, are immunoreactive with Ki-S2 and withantibodies directed against p16^(INK4a). So, in contrast to dysplasticcells, in metaplasias, no cells are double stained using the Ki-S2 andp16^(INK4a) specific antibodies.

These results show that double staining of cells with reagents specificfor Ki-S2 allows discrimination of p16^(INK4a) over-expressingmetaplastic cells from dysplastic cells.

Example 4 Immunofluorescent Detection of the Over-Expression ofp16^(INK4a), Ki67 and PCNA in Bronchioalveolar-Lavage Samples ofIndividuals with Diagnosed Small Cell Lung Cancer (Double Staining)

Cells contained in bronchioalveolar lavage specimens of patients areprepared according to ThinPrep technology. Merckofix®-fixed cytologicalsamples of the lavages of patients diagnosed with small cell lung cancerare immunofluorescent stained using antibodies specific for p16^(INK4a),Ki67, and PCNA.

In this experiment, a procedure is used that does not discriminatebetween staining originating from immunoreactivity against the twoproliferation markers PCNA and Ki67. Within the context of the presentinvention, whether one or the other proliferation marker is expressed inthe cells must not necessarily be answered. The main question is whetherany proliferation marker is expressed. In the course of the experiments,a procedure is chosen that gives the same fluorescence signal as anindication of the presence of a proliferation marker. This procedure issuitable to improve sensitivity of the detection of the cellproliferation characteristics. As necessary, the procedure may beapplied so as to render one detectable signal for three, four, or evenmore marker molecules characteristic of cell proliferation. Analogously,the same may under certain circumstances be true for the INK4a geneexpression products. It must be understood that different stainingsignals for different proliferation marker molecules may be desirable.The procedures may be applied to the necessities of the respectiveexperiment.

The tissue sections are rehydrated through incubation in xylene andgraded ethanol, and then transferred to Aqua bidest. Conventional smearsand liquid-based cytological samples (ThinPreps®) are rehydrated inethanol (50%) for 10 min, and transferred in Aqua bidest. Antigenretrieval is carried out with 10 mM citrate buffer (pH 6.0) forp16^(INK4a), Ki67, and PCNA. Therefore, the slides are heated in awaterbath for 40 min at 95-98° C. The slides are cooled down to RT for20 minutes, transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl,0.05% Tween 20/DakoCytomation: code no.: S3006), and finally, thesamples are surrounded with a lipid-pencil.

To avoid non-specific binding of the secondary antibody (species: goat),the specimens are incubated with 10% goat serum for 30 min at RT.

The slides are then incubated with the primary antibodies, mouseanti-human p16^(INK4a) antibody (3.48 μg/ml), rabbit anti-Ki67, andrabbit anti-PCNA (each 1:25) for 30 min at RT. The slides are thenrinsed with washing buffer and placed in a fresh buffer bath for 5 min.Excess buffer is tapped off, and the specimen is covered with 200 μl ofthe secondary reagent (containing goat anti-mouse antibody,AlexaFluor®488 conjugated and goat anti rabbit antibody and, AlexaFluor®546 conjugated) and then incubated for 30 min at RT. Then slidesare washed two times as before and directly mounted with a specialmounting medium for fluorescence.

Microscopic examination of the slides reveals that cells immunoreactivewith p16^(INK4a) and also with Ki67 or PCNA may be identifiedmicroscopically as cells of small cell lung cancer. Cells stained by thep16^(INK4a)-specific reaction, originating from metaplasias, are notstained by the reaction specific for Ki67 and PCNA. The microscopicinspection of the cell proliferation marker staining shows thatmetaplastic cells over-expressing p16^(INK4a) are not immunoreactivewith antibodies directed against Ki67 and PCNA. Samples containingdysplastic cells, in contrast, comprise cells, that are immunoreactivewith Ki67/PCNA and with antibodies directed against p16^(INK4a). So, incontrast to dysplasias, in metaplasias, no cells may be triple-stainedusing the Ki67, PCNA, and p16^(INK4a) specific antibodies.

These results show that the triple staining of cells with reagentsspecific for Ki67/PCNA allows discrimination of p16^(INK4a)over-expressing non-dysplastic cells from dysplasias.

Example 5 Flow Cytometric Detection of Dysplastic Cells by SimultaneousDetection of mcm5 mRNA, p14ARF Protein, and Ki67 Protein in Cells ofCervical Origin

Cytological samples (Cell suspensions in PBS, pH 7.4) of the cervixuteri were fluorescent stained using antibodies specific for p14ARF andKi-67 and oligoprobes for mcm-5 and evaluated by three-color fluorescentFACS analysis.

Cells were centrifuged, the supernatant decanted and fixed andpermeabilized with 100 ml Permeafix (Ortho Diagnostic, Raitan, N.J.,USA) for 1 hour at room temperature. Cells were washed in sterile PBS,pH 7.4, pelleted, and re-suspended in 100 ml Permeafix for 1 hour atroom temperature. Cells were washed in sterile PBS, pH 7.4, pelleted,and re-suspended in sterile PBS. They were incubated with PE-conjugatedanti-p14ARF antibody and PE-Cy5-conjugated anti-Ki67 antibody for 1 h at+4° C. Cells were washed in sterile PBS, pH 7.4, pelleted, andre-suspended in 100 ml Permeafix for 30 min at room temperature. Cellswere washed in sterile PBS, pelleted by centrifugation, and then washedagain in 2× standard saline citrate (SSC). After centrifugation, thecell pellet was resuspended in hybridization solution (2×SSC, 30%formamide, sonicated salmon sperm, and yeast transfer DNA) containing500 ng of 5-carboxy-fluorescein double end-labelled, mcm5-specificoligonucleotides probes. The intercellular hybridization was performedat 42° C. for 1 hour, followed by successive washes in 2×SSC, 0.5%Triton X-100, and 1×SSC, 0.5% Triton X-100 at 42° C. The cells werere-suspended for analysis in PBS, pH 8.3, and analyzed on a flowcytometer (FACScan, Becton Dickinson, IS). For each analysis,30,000-100,000 gated events were collected. Data analysis was performedusing CellQuest (Becton Dickinson, IS).

The flow cytometer analysis revealed that cells immunoreactive withp14ARF and Ki-67, and/or reactive for the oligoprobes of mcm5, could beidentified only in samples of patients with dysplastic lesions of thecervix. Samples from women with no dysplastic lesions showed noconcomitant staining of p14ARF with Ki67 or mcm5.

These results show that the double-or-triple staining of cells withreagents specific for Ki67 and/or mcm5 allows discrimination of p14ARFover-expressing non-dysplastic cells from p14ARF over-expressingdysplastic cells.

Example 6 Immunoenzymatic Detection of the Over-Expression ofp16^(INK4a) and Ki67 in Histological Samples of the Uterine Cervix(Sequential Double Staining)

Sections of formalin-fixed, paraffin-embedded tissue samples of thecervix uteri were immunoenzymatically double-stained using antibodiesspecific for p16^(INK4a) and Ki67.

The tissue sections were rehydrated through incubation in xylene andgraded ethanol, and transferred to Aqua bidest. Antigen retrieval wascarried out with 10 mM citrate buffer (pH 6.0) for p16^(INK4a) and Ki67.Therefore, the slides were heated in a waterbath for 40 min at 95-98° C.The slides were cooled down to RT for 20 minutes and transferred towashing buffer (DakoCytomation).

Endogenous peroxidase activities were blocked with 3% H₂O₂(DakoCytomation) for 5 min at RT.

After washing the slides for 5 min at RT, they were incubated with thefirst primary antibody, mouse anti-human p16^(INK4a) antibody (MTM) for30 min at RT, and were then rinsed with washing buffer and placed in afresh buffer bath for 5 min. Excess buffer was tapped off and eachspecimen was covered with 200 μl of the secondary reagent (EnVision goatanti mouse—Peroxidase/DakoCytomation), and incubated for 30 min at RT.Then slides were washed three times as before. For the chromogenicvisualization, DAB (DakoCytomation) was used by incubating the slideswith the substrate chromogen complex for 10 min at RT. The reaction wasstopped in deionized water, and the slides placed in wash buffer.

After washing, the slides were incubated with the second primaryantibody, rabbit anti-human Ki67 antibody (Dianova, clone Ab-3) for 30min at RT, and were then rinsed with washing buffer and placed in afresh buffer bath for 5 min. Excess buffer was tapped off, and eachspecimen was covered with 200 μl of the secondary reagent (goat antirabbit—Alkaline Phosphatase labeled/DakoCytomation) and incubated for 30min at RT. Then slides were washed three times as before. For thechromogenic visualization, FastRed (BioGenex) was used by incubating theslides with the substrate chromogen complex for 30 min at RT. Thereaction was stopped in deionized water.

After counterstaining with hematoxylin (DakoCytomation) for 2 min at RT,the slides were incubated in running tap water for 10 min at RT, andthen mounted with aqueous mounting medium (Aquatex/MERCK).

The microscopic examination of the slides revealed that cellsimmunoreactive with p16^(INK4a) and Ki67 were found only in samples thatwere identified microscopically as samples of dysplastic lesions. Cellsstained by the p16^(INK4a)-specific reaction, originating frommetaplasias, were not stained by the reaction specific for Ki67.Microscopic inspection of the cell proliferation marker staining showedthat metaplastic cells over-expressing p16^(INK4a) were notimmunoreactive with antibodies directed against Ki67. Samples containingdysplastic tissue areas, in contrast, comprised cells that wereimmunoreactive with Ki67 and with antibodies directed againstp16^(INK4a). So, in contrast to dysplasias, in metaplasias, no cellswere double-stained using the Ki67 and p16^(INK4a) specific antibodies.

These results (FIG. 7) show that double staining of cells with reagentsspecific for Ki67 and p16^(INK4a) also produces a specificdouble-staining pattern in chromogenic staining procedures.

Example 7 Immunoenzymatic Detection of the Over-Expression ofp16^(INK4a) and Ki67 in Cytological Samples of the Uterine Cervix(Sequential Double Staining)

Merckofix® fixed cytological samples (conventional smears andliquid-based cytology (ThinPreps®)) of the cervix uteri wereimmunoenzymatically double-stained using antibodies specific forp16^(INK4a) and Ki67.

Conventional smears and liquid-based cytological samples were rehydratedin ethanol (50%) for 10 min at RT and transferred in Aqua bidest.Antigen retrieval was carried out with 10 mM citrate buffer (pH 6.0) forp16^(INK4a) and Ki67. Therefore, the slides were heated in a waterbathfor 40 min at 95-98° C. The slides were cooled down to RT for 20 minutesand transferred to washing buffer.

Endogenous peroxidase activities were blocked with 3% H₂O₂ for 5 min atRT.

After washing, the slides were incubated with the first primaryantibody, mouse anti-human p16^(INK4a) antibody, for 30 min at RT, andwere then rinsed with washing buffer and placed in a fresh buffer bathfor 5 min. Excess buffer was tapped off, and each specimen was coveredwith 200 μl of the secondary reagent (EnVision goat antimouse—Peroxidase) and incubated for 30 min at RT. Then the slides werewashed three times as before. For the chromogenic visualization, DAB(DakoCytomation) was used by incubating the slides with the substratechromogen complex for 10 min at RT. The reaction was stopped indeionized water, and the slides placed in wash buffer.

After washing, the slides were incubated with the second primaryantibody, rabbit anti-human Ki67 antibody (Dianova, clone Ab-3) for 30min at RT, and were then rinsed with washing buffer and placed in afresh buffer bath for 5 min. Excess buffer was tapped off, and eachspecimen was covered with 200 μl of the secondary reagent (goat antirabbit—Alkaline Phosphatase labeled/DakoCytomation) and incubated for 30min at RT. Then the slides were washed three times as before. For thechromogenic visualization, Fast Red (BioGenex) was used by incubatingthe slides with the substrate chromogen complex for 30 min at RT. Thereaction was stopped in deionized water.

After counterstaining with hematoxylin (DakoCytomation) for 2 min at RT,the slides were incubated in running tap water for 10 min at RT, andthen mounted with aqueous mounting medium (Aquatex/MERCK).

Microscopic examination of the slides revealed that cells immunoreactivewith p16^(INK4a) and Ki67 could be identified as dysplastic cells on thebasis of their morphology. Cells stained by the p16^(INK4a)-specificreaction originating from metaplasias were not stained by the reactionspecific for Ki67. The microscopic inspection of the cell proliferationmarker staining showed that metaplastic cells over-expressingp16^(INK4a) were not immunoreactive with the antibodies directed againstKi67. Dysplastic cells, in contrast, were immunoreactive with Ki67 andwith antibodies directed against p16^(INK4a). So, in contrast tometaplastic cells, in dysplastic cells, double-staining of single cellscould be produced using the Ki67 and p16^(INK4a) specific antibodies.

These results (FIG. 8) show that the double staining of cells withreagents specific for Ki67 and p16^(INK4a) also produces a specificdouble staining pattern in chromogenic staining procedures.

1. A method for discriminating dysplastic cells non-dysplastic cells ina biological sample comprising determining in the biological sample theco-expression of at least two marker molecules in at least one singlecell, wherein at least one marker molecule is an INK4a gene product,which is an expression product encoded by the INK4a gene, and at leastone further marker molecule is a cell proliferation marker, wherein theover-expression of the at least one INK4a gene product and expression ofthe at least one cell proliferation marker for active cell proliferationat a detectable level within said single cell is indicative of thedysplastic state of the cell, and the over-expression of the at leastone INK4a gene product and expression of the at least one cellproliferation marker for senescence, terminal cell differentiation,apoptosis or cell cycle arrest at a detectable level within said singlecell is indicative of the non-dysplastic state of the cell.
 2. Themethod according to claim 1, wherein a set of two or more cellproliferation markers is detected.
 3. The method according to claim 1,wherein the INK4a gene product has a molecular weight between 13 and 19kDa.
 4. The method according to claim 1, wherein the INK4a gene productis selected from the group consisting of p16^(INK4a) and p14ARF.
 5. Themethod according to claim 1, wherein the cell proliferation marker isselected from the group consisting of a proliferation marker necessaryfor the maintenance of cell proliferation, a proliferation markerengaged in DNA replication, a proliferation marker being or encoding amember of the processive replication fork, a senescence marker, a cellcycle arrest marker and an apoptosis marker.
 6. The method according toclaim 5, wherein the proliferation marker necessary for the maintenanceof cell proliferation is a molecule selected from the group consistingof Ki67 molecules, Ki-S5 molecules and Ki-S2 molecules.
 7. The methodaccording to claim 6, wherein the proliferation marker engaged in DNAreplication is selected from the group consisting of helicases orsubunits thereof, cell division cycle molecules, phosphatase moleculesand kinase molecules.
 8. The method according to claim 7, wherein thehelicases or subunits thereof are selected from the group consisting ofMCM2, MCM3, MCM4, MCM5, MCM6, MCM7 and HELAD1.
 9. The method accordingto claim 7, wherein the cell division cycle molecules, kinases moleculesand phosphatases molecules are selected from the group consisting ofCDC6, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 andMCM10.
 10. The method according to claim 5, wherein the member of theprocessive replication fork is selected from the group consisting ofPCNA and POLD.
 11. The method according to claim 1, wherein the INK4agene product is a polypeptide or a nucleic acid molecule.
 12. The methodaccording to claim 1, wherein additionally at least one further markermolecule is detected for improvement of the assessment of diagnosis orprognosis.
 13. The method according to claim 12, wherein the furthermarker molecule is at least one further proliferation marker molecule.14. The method according to claim 12, wherein the further markermolecule is selected from the group consisting of a senescence marker,an apoptosis marker, a cell cycle arrest marker, a marker for terminaldifferentiation of cells, a marker for viral infection, a marker forviral activity, a cell cycle regulatory protein, a gene-productnecessary for the maintenance of cell proliferation, a gene-productengaged in DNA replication, and a gene product being a member of theprocessive replication fork.
 15. The method according to claim 12,wherein additionally a cytological staining procedure employing at leastone dye selected from the group consisting of DAPI, Quinacrin,Chromomycin, Azan, Acridin-orange, Hematoxylin, Eosin, Sudan-red,Toluidin-blue, and Thionin, or a staining method selected from the groupconsisting of Pap-staining, Giemsa-staining, Hematoxylin-Eosin staining,van-Gieson-staining, Schiff-staining, staining via metal precipitates,Turnbulls-blue-staining and staining via metal cyanides, is applied. 16.The method according to claim 1, wherein the dysplastic cells are cellsof a cancerous or precancerous lesion.
 17. The method according to claim16, wherein the dysplastic cells are cells of a dysplasia beingassociated with a papilloma virus.
 18. The method according to claim 17,wherein the papilloma virus is a high risk human papilloma virusselected from the group consisting of HPV16, HPV18, HPV31, HPV 33,HPV35, HPV 39, HPV 45, HPV 51, HPV 52, HPV56, HPV 58, HPV 59, HPV 66 andHPV
 68. 19. The method according to claim 16, wherein the lesion isselected from the group consisting of a lesion of the anogenital tract,a lesion of the respiratory tract and a lesion of the skin and it'sappendages.
 20. The method according to claim 19, wherein the lesion isselected from the group consisting of a lesion of the uterine cervix, alesion of the vagina, a lesion of the vulva, a lesion of the penis, alesion of the anus, a lesion of the rectum, a lesion of the bronchictree, a lesion of the lung, a lesion of the peritoneal space, a lesionof the naso-pharyngeal space, a lesion of the oral cavity of and alesion of the skin.
 21. The method according to claim 1, wherein thebiological sample is a sample containing cells originating from theanogenital tract, from the respiratory tract or from the skin and itsappendages.
 22. The method according to claim 21, wherein the biologicalsample is obtained from the uterine cervix, the vagina, the vulva, thepenis, the anus, the rectum, the bronchic tree, the lung, thenaso-pharyngeal space, the oral cavity or the skin.
 23. The methodaccording to claim 22, wherein the biological sample is a cytological orhistological preparation.
 24. The method according to claim 1, whereinthe determination of the 14a gene product and/or the cell proliferationmarker molecules is performed using at least one probe specificallyrecognising at least one of the respective molecules to be detected. 25.The method according to claim 24, wherein the at least one probe isdetectably labelled with at least one label.
 26. The method according toclaim 25, wherein the label is selected from the group consisting of aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and an enzyme.
 27. The methodaccording to claim 24, wherein the probe is a protein and/or a nucleicacid.
 28. The method according to claim 27, wherein the protein is anantibody directed against a INK4a encoded gene product or a cellproliferation marker gene product.
 29. The method according to claim 28,which comprises an immuno-cytochemical staining procedure.
 30. Themethod according to claim 25, wherein the probe is a nucleic acidspecifically hybridizing to an INK4a gene product or a cellproliferation marker gene product.
 31. The method according to claim 30,which comprises an in situ hybridization reaction.
 32. The methodaccording to claim 30, which comprises a nucleic acid amplificationreaction.
 33. The method according to claim 32, wherein the nucleic acidamplification reaction is PCR, NASBA or LCR.
 34. The method according toclaim 27, wherein the determination using nucleic acid probes andpolypeptide probes are carried out simultaneously.
 35. A kit comprisingat least one or more probes for the detection of the presence or absenceand/or the level of the over-expression of at least one INK4a geneproduct and at least one cell proliferation marker gene product inbiological samples.
 36. The kit according to claim 35, wherein the INK4agene products are selected from the group consisting of p16^(INK4a) andp14ARF.
 37. The kit according to claim 35, wherein the cellproliferation marker gene products are selected the group consisting ofCDC6, MCM3, MCM3, MCM4, MCM5, MCM6, MCM7, CDC7 protein kinase, Dbf4,CDC14 protein phosphatase, CDC45 and MCM10, Ki67, Ki-S2, PCNA and POLD.38. The kit according to claim 35 furthermore comprising at least one ofthe following: a. a p16^(INK4a) sample for carrying out a positivecontrol reaction, b. a p14ARF sample for carrying out a positive controlreaction, c. a Ki67 sample for carrying out a positive control reaction,d. a Ki-S2 sample for carrying out a positive control reaction, e. anMCM5 sample for carrying out a positive control reaction, f. an MCM2sample for carrying out a positive control reaction, g. a PCNA samplefor carrying out a positive control reaction, h. reagents for detectionof the presence or absence and/or the level of p16^(INK4a), i. reagentsfor detection of the presence or absence and/or the level of p14ARF, j.reagents for detection of the presence or absence and/or the level ofKi67. k. reagents for detection of the presence or absence and/or thelevel of Ki-S2. l. reagents for detection of the presence or absenceand/or the level of MCM5. m. reagents for detection of the presence orabsence and/or the level of MCM2 n. reagents for detection of thepresence or absence and/or the level of PCNA, o. one or more samples ofINK4a gene-products for carrying out positive control reactions, p. oneor more samples of cell proliferation marker gene-products for carryingout positive control reactions, q. one or more reagents for thedetection of the presence or absence and/or the level of other INK4agene products, or r. and one or more reagents for the detection of thepresence or absence and/or the level of other cell proliferation markergene products.